px458 camta1 sgrna  (Addgene inc)

Journal: bioRxiv

Article Title: Sequential transcriptional gates in the thalamo-cortical circuit coordinate memory stabilization

doi: 10.1101/2025.05.28.656415

Figure Lengend Snippet: a , Average expression of all transcriptional regulators in ANT neurons across conditions and time points (units log 2 CPM +1). b , Average expression of all transcriptional regulators in ACC neurons across conditions and time points (units log 2 CPM +1). c , Line plots of average expression of ANT (left) and ACC (right) transcriptional regulators in HR neurons only across behavioral time points (units log 2 CPM +1). d , Relative expression of pseudo-time signature genes in ANT at early (top) and remote retrieval (bottom) between HR and LR samples, normalized to Map2 , n = 3-6 biological replicates, * P = 0.0211 for Camta1 ; * P = 0.0495 for Gria2 ; * P = 0.0286 for Clu , * P = 0.0384 for Cadm1, unpaired t-test, mean± SEM shown. e , Relative expression of late pseudo-time signature genes in ACC between HR and LR samples, normalized to Map2 , n = 5-6 biological replicates, mean± SEM shown.

Article Snippet: Briefly, 1×10^ Neuro2A cells were transfected with Px458 -Tcf4- sgRNA (Addgene #48138) or Px458- Camta1- sgRNA (Addgene #48138) for 72 hours and harvested.

Techniques: Expressing

Journal: bioRxiv

Article Title: Sequential transcriptional gates in the thalamo-cortical circuit coordinate memory stabilization

doi: 10.1101/2025.05.28.656415

Figure Lengend Snippet: a , Schematic of in vitro CRISPR screen workflow. b , Neuro 2A cells transfected with sgRNA-spCas9-GFP, scale: 200um. c , Stacked bar plot of wildtype (WT) vs mutant percent reads of amplicon target regions from DNA collected from Neuro 2A cells post-transfection. d , Breakdown of Cas9-induced indel mutations in Neuro 2A cells expressing a sgRNA targeting one of the TRs. e , Top left: coronal slice from a Camta1 -KO Rosa26-Cas9 mouse in ANT. Top middle inserts: 10X field of view from Camta1 -KO displaying DAPI, CAMTA1 protein and sgRNA expression. Top right: 10X field of view from a control Rosa26-Cas9 mouse injected with CRE recombinase. Bottom left: coronal slice from a Creb1 -KO in HPC. Bottom middle inserts: 10X field of view from Creb1 -KO displaying DAPI, CREB1 protein and sgRNA expression. Bottom right: 10X field of view from a control Rosa26-Cas9 mouse injected with CRE recombinase. f , Immunofluorescent staining of NeuN or Cleaved Caspase 3 in Rosa26-Cas9 mouse injected with Creb1 -sgRNA to evaluate extent of neuronal toxicity from AAV injection. Scale: 100µm in 4X, 50µm in 10X. g , Western blot validation of Tcf4 -KO from ANT tissue compared to control animal. h , Discrimination indices for learning and recall performances in HR of Rosa26LSL-spCas9-eGFP mice expressing sgRNA targeting Myt1l , Mef2c or Kmt2a ( n = 6-7 mice per cohort), individual data points shown (faded lines), with mean ± SEM (solid line). g , Example traces of F(z) from one control mouse on late training from ACC and ANT aligned to lick rate and task zones, three trial shown.

Article Snippet: Briefly, 1×10^ Neuro2A cells were transfected with Px458 -Tcf4- sgRNA (Addgene #48138) or Px458- Camta1- sgRNA (Addgene #48138) for 72 hours and harvested.

Techniques: In Vitro, CRISPR, Transfection, Mutagenesis, Amplification, Expressing, Control, Injection, Staining, Western Blot, Biomarker Discovery

Journal: bioRxiv

Article Title: Sequential transcriptional gates in the thalamo-cortical circuit coordinate memory stabilization

doi: 10.1101/2025.05.28.656415

Figure Lengend Snippet: a , Schematic of the CRISPR-Cas9 screen workflow. b , Stacked bar plot of WT vs mutant reads of amplicon regions from Rosa26LSL-spCas9-eGFP mice injected with AAV-sgRNA targeting the TRs shown. c , Breakdown of Cas9-induced indel mutations. d , Top: timeline of in vivo CRISPR-Cas9 behavioral screen, crossed pattern block represent AAV-sgRNA injection, and squared pattern block is window of behavioral testing. Below: discrimination indices for learning and recall performances in HR for mice expressing AAV- Cre as control or AAV-sgRNA in HPC, ANT or ACC, n = 7-9 mice per cohort, individual data points shown (faded lines), with mean ± SEM (solid line), * P = 0.048 between controls and sgRNA- creb1 on T6, * P = 0.026 on T8, ** P = 0.0063 on mid retrieval and *** P = 0.0003 on remote retrieval; * P = 0.0288 between controls and sgRNA- camta1 on mid retrieval and * P = 0.0466 on remote retrieval; *** P = 0.0002 between controls and sgRNA- tcf4 on remote retrieval; ** P = 0.0086 between controls and sgRNA- ash1l on remote retrieval, One-way ANOVA with Bonferroni correction. e, f , Discrimination indices for mice injected with sgRNA- camta1 in ACC ( e ) or sgRNA- ash1l in ANT ( f ). g , Discrimination indices for learning and recall performances in HR for photometry cohort, n = 7-10 mice, * P = 0.0248 between controls and sgRNA- camta1 on mid retrieval and ** P = 0.0031 on remote retrieval; * P = 0.0306 between controls and sgRNA- tcf4 on remote retrieval, One-way ANOVA with Bonferroni correction. h , Left: sample entropy of all trials during cue of HR retrieval sessions in ANT, * P = 0.0361 between controls and sgRNA- tcf4 on mid retrieval and * P = 0.0221 on remote retrieval. Right: pairwise Pearson’s correlations between ANT and ACC during cue of HR retrieval sessions, at ∼25 trials/mouse. mean ± SEM shown, * P = 0.0163 between controls and sgRNA- camta1 on recent retrieval and * P = 0.0464 on mid retrieval; ** P = 0.0033 between controls and sgRNA- tcf4 on recent retrieval, ** P = 0.0036 on mid retrieval and ** P = 0.0011 on remote retrieval. i, j, Top: scored average expression of Camta1 ( i ) or Tcf4 ( k ) targets derived from ChIP-seq data. Bottom: bar plot of normalized counts in peak regions associated with target genes in control versus knockout samples, n = 1. k, Left: heatmap of H3K4me3 marks in ACC control samples across mid and remote retrieval. Middle: scored average expression of Ash1l targets derived from ChIP-seq data. Right: bar plot of normalized counts in peak regions associated with target genes in control versus Ash1l -knockout samples, n = 2. l , Proposed model for the role of Camta1 , Tcf4 and Ash1l in memory stabilization. Early molecular cascades, such as CREB-dependent, are triggered in the HPC and operate on the scale of hours-days, while expression of Camta1 and Tcf4 in the ANT extend memories beyond days through synaptic plasticity mechanisms. In the ACC, histone methylators, such as Ash1l , operate on longer time constants, allowing the stabilization of information across cortical ensembles.

Article Snippet: Briefly, 1×10^ Neuro2A cells were transfected with Px458 -Tcf4- sgRNA (Addgene #48138) or Px458- Camta1- sgRNA (Addgene #48138) for 72 hours and harvested.

Techniques: CRISPR, Mutagenesis, Amplification, Injection, In Vivo, Blocking Assay, Expressing, Control, Derivative Assay, ChIP-sequencing, Knock-Out

Journal: bioRxiv

Article Title: Sequential transcriptional gates in the thalamo-cortical circuit coordinate memory stabilization

doi: 10.1101/2025.05.28.656415

Figure Lengend Snippet: a , Example traces of F(z) from one control mouse from late training form ACC and ANT aligned to lick rate and task zones, three trials shown. b , Schematic of ChIP-Seq workflow: selection and validation of antibodies via generation of knockout N2A cell lines, followed by ChIP seq of animals collected from recent, mid, remote retrieval (for WT and Ash1l KO, H3K4me3 antibody) or from mid-retrieval (WT and Camta1 / Tcf4 KO). c , Tornado plots of overall ChIP-seq signal for controls and Camta1 / Tcf4 KO. d , Top: ChIP-seq signal at gene loci for Camta1 (yellow) in WT vs. Camta1 -KO mice at mid retrieval. Bottom: ChIP-seq signal at gene loci for Tcf4 (red) in WT vs. Tcf4 -KO mice at mid retrieval. Tracks show normalized read density (RPKM) and are color-coded by condition (darker shade for wildtype, lighter shade for KO). e , Gene ontology analysis derived from ChIP-seq differential peak analysis of WT vs. Camta1 -KO (yellow) or WT vs. Tcf4 -KO (red) at mid retrieval. f , Gene ontology analysis derived from H3K4me3 peaks (accessible chromatin) in wildtype animals at mid or remote retrieval. g , ChIP-seq signal at gene loci for H3K4me3 in WT or Ash1l -KO mice at remote retrieval. Tracks show normalized read density (RPKM) and are color-coded by condition (darker shade for wildtype, lighter shade for Ash1l -KO). h, Gene ontology analysis derived from H3K4me3 peaks from Ash1l -KO mice at mid and remote retrieval. i , Bar plots of downregulation of plasticity related gene targets in Camta1 /-KO versus control animals (yellow), * P = 0.00159 for Arp21; ** P = 0.0018 for Rasgrp1; * P = 0.0231 for Snap25, n = 4 biological replicates, errors bars are SEM.

Article Snippet: Briefly, 1×10^ Neuro2A cells were transfected with Px458 -Tcf4- sgRNA (Addgene #48138) or Px458- Camta1- sgRNA (Addgene #48138) for 72 hours and harvested.

Techniques: Control, ChIP-sequencing, Selection, Biomarker Discovery, Knock-Out, Derivative Assay

Journal: bioRxiv

Article Title: Sequential transcriptional gates in the thalamo-cortical circuit coordinate memory stabilization

doi: 10.1101/2025.05.28.656415

Figure Lengend Snippet: a , Schematic of in vitro CRISPR screen workflow. b , Neuro 2A cells transfected with sgRNA-spCas9-GFP, scale: 200um. c , Stacked bar plot of wildtype (WT) vs mutant percent reads of amplicon target regions from DNA collected from Neuro 2A cells post-transfection. d , Breakdown of Cas9-induced indel mutations in Neuro 2A cells expressing a sgRNA targeting one of the TRs. e , Top left: coronal slice from a Camta1 -KO Rosa26-Cas9 mouse in ANT. Top middle inserts: 10X field of view from Camta1 -KO displaying DAPI, CAMTA1 protein and sgRNA expression. Top right: 10X field of view from a control Rosa26-Cas9 mouse injected with CRE recombinase. Bottom left: coronal slice from a Creb1 -KO in HPC. Bottom middle inserts: 10X field of view from Creb1 -KO displaying DAPI, CREB1 protein and sgRNA expression. Bottom right: 10X field of view from a control Rosa26-Cas9 mouse injected with CRE recombinase. f , Immunofluorescent staining of NeuN or Cleaved Caspase 3 in Rosa26-Cas9 mouse injected with Creb1 -sgRNA to evaluate extent of neuronal toxicity from AAV injection. Scale: 100µm in 4X, 50µm in 10X. g , Western blot validation of Tcf4 -KO from ANT tissue compared to control animal. h , Discrimination indices for learning and recall performances in HR of Rosa26LSL-spCas9-eGFP mice expressing sgRNA targeting Myt1l , Mef2c or Kmt2a ( n = 6-7 mice per cohort), individual data points shown (faded lines), with mean ± SEM (solid line). g , Example traces of F(z) from one control mouse on late training from ACC and ANT aligned to lick rate and task zones, three trial shown.

Article Snippet: Briefly, 1×10^ Neuro2A cells were transfected with Px458 -Tcf4- sgRNA (Addgene #48138) or Px458- Camta1- sgRNA (Addgene #48138) for 72 hours and harvested.

Techniques: In Vitro, CRISPR, Transfection, Mutagenesis, Amplification, Expressing, Control, Injection, Staining, Western Blot, Biomarker Discovery

Journal: bioRxiv

Article Title: Sequential transcriptional gates in the thalamo-cortical circuit coordinate memory stabilization

doi: 10.1101/2025.05.28.656415

Figure Lengend Snippet: a , Schematic of the CRISPR-Cas9 screen workflow. b , Stacked bar plot of WT vs mutant reads of amplicon regions from Rosa26LSL-spCas9-eGFP mice injected with AAV-sgRNA targeting the TRs shown. c , Breakdown of Cas9-induced indel mutations. d , Top: timeline of in vivo CRISPR-Cas9 behavioral screen, crossed pattern block represent AAV-sgRNA injection, and squared pattern block is window of behavioral testing. Below: discrimination indices for learning and recall performances in HR for mice expressing AAV- Cre as control or AAV-sgRNA in HPC, ANT or ACC, n = 7-9 mice per cohort, individual data points shown (faded lines), with mean ± SEM (solid line), * P = 0.048 between controls and sgRNA- creb1 on T6, * P = 0.026 on T8, ** P = 0.0063 on mid retrieval and *** P = 0.0003 on remote retrieval; * P = 0.0288 between controls and sgRNA- camta1 on mid retrieval and * P = 0.0466 on remote retrieval; *** P = 0.0002 between controls and sgRNA- tcf4 on remote retrieval; ** P = 0.0086 between controls and sgRNA- ash1l on remote retrieval, One-way ANOVA with Bonferroni correction. e, f , Discrimination indices for mice injected with sgRNA- camta1 in ACC ( e ) or sgRNA- ash1l in ANT ( f ). g , Discrimination indices for learning and recall performances in HR for photometry cohort, n = 7-10 mice, * P = 0.0248 between controls and sgRNA- camta1 on mid retrieval and ** P = 0.0031 on remote retrieval; * P = 0.0306 between controls and sgRNA- tcf4 on remote retrieval, One-way ANOVA with Bonferroni correction. h , Left: sample entropy of all trials during cue of HR retrieval sessions in ANT, * P = 0.0361 between controls and sgRNA- tcf4 on mid retrieval and * P = 0.0221 on remote retrieval. Right: pairwise Pearson’s correlations between ANT and ACC during cue of HR retrieval sessions, at ∼25 trials/mouse. mean ± SEM shown, * P = 0.0163 between controls and sgRNA- camta1 on recent retrieval and * P = 0.0464 on mid retrieval; ** P = 0.0033 between controls and sgRNA- tcf4 on recent retrieval, ** P = 0.0036 on mid retrieval and ** P = 0.0011 on remote retrieval. i, j, Top: scored average expression of Camta1 ( i ) or Tcf4 ( k ) targets derived from ChIP-seq data. Bottom: bar plot of normalized counts in peak regions associated with target genes in control versus knockout samples, n = 1. k, Left: heatmap of H3K4me3 marks in ACC control samples across mid and remote retrieval. Middle: scored average expression of Ash1l targets derived from ChIP-seq data. Right: bar plot of normalized counts in peak regions associated with target genes in control versus Ash1l -knockout samples, n = 2. l , Proposed model for the role of Camta1 , Tcf4 and Ash1l in memory stabilization. Early molecular cascades, such as CREB-dependent, are triggered in the HPC and operate on the scale of hours-days, while expression of Camta1 and Tcf4 in the ANT extend memories beyond days through synaptic plasticity mechanisms. In the ACC, histone methylators, such as Ash1l , operate on longer time constants, allowing the stabilization of information across cortical ensembles.

Article Snippet: Briefly, 1×10^ Neuro2A cells were transfected with Px458 -Tcf4- sgRNA (Addgene #48138) or Px458- Camta1- sgRNA (Addgene #48138) for 72 hours and harvested.

Techniques: CRISPR, Mutagenesis, Amplification, Injection, In Vivo, Blocking Assay, Expressing, Control, Derivative Assay, ChIP-sequencing, Knock-Out

Journal: bioRxiv

Article Title: Sequential transcriptional gates in the thalamo-cortical circuit coordinate memory stabilization

doi: 10.1101/2025.05.28.656415

Figure Lengend Snippet: a , Example traces of F(z) from one control mouse from late training form ACC and ANT aligned to lick rate and task zones, three trials shown. b , Schematic of ChIP-Seq workflow: selection and validation of antibodies via generation of knockout N2A cell lines, followed by ChIP seq of animals collected from recent, mid, remote retrieval (for WT and Ash1l KO, H3K4me3 antibody) or from mid-retrieval (WT and Camta1 / Tcf4 KO). c , Tornado plots of overall ChIP-seq signal for controls and Camta1 / Tcf4 KO. d , Top: ChIP-seq signal at gene loci for Camta1 (yellow) in WT vs. Camta1 -KO mice at mid retrieval. Bottom: ChIP-seq signal at gene loci for Tcf4 (red) in WT vs. Tcf4 -KO mice at mid retrieval. Tracks show normalized read density (RPKM) and are color-coded by condition (darker shade for wildtype, lighter shade for KO). e , Gene ontology analysis derived from ChIP-seq differential peak analysis of WT vs. Camta1 -KO (yellow) or WT vs. Tcf4 -KO (red) at mid retrieval. f , Gene ontology analysis derived from H3K4me3 peaks (accessible chromatin) in wildtype animals at mid or remote retrieval. g , ChIP-seq signal at gene loci for H3K4me3 in WT or Ash1l -KO mice at remote retrieval. Tracks show normalized read density (RPKM) and are color-coded by condition (darker shade for wildtype, lighter shade for Ash1l -KO). h, Gene ontology analysis derived from H3K4me3 peaks from Ash1l -KO mice at mid and remote retrieval. i , Bar plots of downregulation of plasticity related gene targets in Camta1 /-KO versus control animals (yellow), * P = 0.00159 for Arp21; ** P = 0.0018 for Rasgrp1; * P = 0.0231 for Snap25, n = 4 biological replicates, errors bars are SEM.

Article Snippet: Briefly, 1×10^ Neuro2A cells were transfected with Px458 -Tcf4- sgRNA (Addgene #48138) or Px458- Camta1- sgRNA (Addgene #48138) for 72 hours and harvested.

Techniques: Control, ChIP-sequencing, Selection, Biomarker Discovery, Knock-Out, Derivative Assay

Journal: bioRxiv

Article Title: Selective RNA sequestration in biomolecular condensates directs cell fate transitions

doi: 10.1101/2025.05.08.652299

Figure Lengend Snippet: (a) mRNA targets of 53 RNA-binding proteins were analyzed for their enrichment in P-bodies and cytoplasm, using CLIP-seq experiments available in the CLIPdb 1.0 database. Circle size indicates counts; color indicates p-value. (b) UMAP analysis of small RNA-seq data for the indicated samples based on differentially expressed genes between purified P-body and cytoplasmic fractions. (c) Heatmap showing expression levels of differentially enriched miRNAs between purified P-body and cytoplasmic fractions in mouse naïve ES cells. (n=2, p < 0.05). (d) Quantification of P-body enrichment for miRNA targets and their corresponding translation efficiency (TE). Ribosome profiling data from1. (e, f) Box plots showing P-body enrichment of Let-7 (e) and miR-300/381 targets (f) in naïve and primed mouse ES cells and mouse fibroblasts. Unpaired t-test ± s.d, showing Holm’s method adjusted p-values, ****p<0.0001. (g) Box plot comparing P-body enrichment of miRNA-targets between WT and AGO2 KO mouse naïve ES cells. Unpaired t-test, showing Holm’s method adjusted p-values, ns: p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (h) Schematic of iPS cell reprogramming, including perturbation of the alternative polyadenylation regulator Nudt21. (i) PolyA site usage index (PDUI) of p-body and cytoplasm enriched transcripts. PDUI<0.5 indicates proximal polyA preference, PDUI>0.5 indicates distal polyA preference Unpaired t-test, showing Holm’s method adjusted p-values. (j) Change in polyadenylation site usage upon Nudt21 KD.

Article Snippet: Briefly, v6.5 mouse ES cells underwent transfection with pX458-sgRNA_Ago2_3/4 plasmids (Addgene # 73531 and 73532).

Techniques: RNA Binding Assay, RNA Sequencing, Purification, Expressing

Journal: bioRxiv

Article Title: Selective RNA sequestration in biomolecular condensates directs cell fate transitions

doi: 10.1101/2025.05.08.652299

Figure Lengend Snippet: (a) Representative western blot showing AGO2 protein levels in WT and AGO2 KO mouse naïve cells. (b) Representative IF images of EDC4 puncta (green) in naïve and primed mouse ES cells. Cell membranes were labeled with Phalloidin (red), and nuclei were counterstained with DAPI (blue) (scale: 10μm) (left panel). P-body number in WT (n=60 cells) and AGO2 KO (n=60 cells) mouse naïve ES cells (right panel). Unpaired Student’s t-test, mean ± s.d., n.s.: p >0.05. (c) Flow cytometry quantification of OCT4-GFP + cells in control and Nudt21 KD reprogramming intermediates at day 8. (d) qRT-PCR analysis of the expression of ES-specific genes in control and Nudt21 KD reprogramming samples. Unpaired Student’s t test, n=3, mean ± s.d., ***: p<0.001, ****: p<0.0001. (e) Representative IF imaging of EDC4 puncta (green) in control and Nudt21 KD reprogramming samples. Nuclei were counterstained with DAPI (blue) (scale: 10μm) and P-body number in control (n=90 cells) and Nudt21 KD induced (dox, n=90 cells) reprogramming samples (right panel). Unpaired Student’s t-test, mean ± s.d., ns: p>0.05. (f) Heatmap showing expression levels of differentially enriched PGCLC-related mRNAs between purified P-body and cytoplasmic fractions in human primed ES cells (n=2). (g) A model showing miRNA-mediated RNA sequestration in cell fate.

Article Snippet: Briefly, v6.5 mouse ES cells underwent transfection with pX458-sgRNA_Ago2_3/4 plasmids (Addgene # 73531 and 73532).

Techniques: Western Blot, Labeling, Flow Cytometry, Control, Quantitative RT-PCR, Expressing, Imaging, Purification